One of the useful tools in immunology is the detection of antigens by enzymes linked to antibodies. This technology employs a substrate which the enzymes process in such a manner as to signal the presence of the antigen if antibodies are present which correspond to the disease for which diagnosis is sought.
A typical and particularly useful procedure of this class is the enzyme immunoassay procedure for the detection of the antibody to human immunodeficiency (AIDS) virus in human blood serum or plasma. This procedure is also known as, and is termed herein, the Western Blot procedure. (Tsang, Victor C. W., Peralta, Jose M., and Simons A. Ray, Methods in Enzymology, Vol. 92, 377-391 (1983). "[29] Enzyme-linked Immunoelectrotransfer Blot Techniques (EITB) for the Study of the Specificities of Antigens and Antibodies separated by Gel Electrophoresis"; see particularly page 377, second paragraph and pages 383-7 for descriptions of the procedure which has become known as the Western Blot procedure.).
Briefly stated, the Western blot procedure takes advantage of the fact that the AIDS and other viruses are constituted by a number of different proteins having different molecular sizes. These can be segregated according to size by subjecting the virus on gel to an applied electric field. Thereupon the proteins migrate distances determined by their molecular weight, thereby segregating them.
In applying the Western blot technique, the segregated proteins are transferred by blotting onto a piece of nitrocellulose or other specifically absorbent paper.
The paper containing the virus protein next is cut into strips and the strips exposed to human blood sera suspected of containing antibodies to the AIDS virus (e.g. "sero-positive" AIDS patients). If antibodies are present, they will adhere specifically to the corresponding viral protein. The strip is washed to remove any material not thus specifically adherent to the viral protein.
The strip mounting the combination of viral protein and antibody next is exposed to a solution containing a second antibody (e.g., goat anti-human antibody), which will bind specifically to human-derived antibodies. To the goat anti-human antibody is chemically (covalently) bound (conjugated) a peroxidase enzyme such as horseradish peroxidase.
The goat antibody thus serves as a carrier for the enzyme and binds it to the viral protein-antibody complex. If the serum under diagnosis in fact contains antibodies to the virus, there thus is formed a complex of AIDS virus protein, serum-derived antibodies to such protein, and goat anti-human antibody to which is chemically bound an enzyme peroxidase. The strip is washed with phosphate-buffered saline solution, or other suitable wash liquid, to remove other materials.
The strip now is exposed to a reagent containing buffered hydrogen peroxide and a chromogen. If the strip contains the enzyme peroxidase, which it can contain only in the event that it also mounts AIDS antibodies, the peroxidase will act with the hydrogen peroxide co-substrate, to peroxidize the chromogen co-substrate, converting it to an intensely dark brown, highly visible product. The oxidized product is insoluble and deposits on the strip precisely where the electrophoretically deposited antibody proteins are located.
The result of this procedure is a blot strip having a characteristic pattern of highly colored bands, if the diagnostic test is positive. No such bands will be present on the strip if the test is negative.
The chromogen-containing reagent thus serves as an indicator for making visible the peroxidase on the strip. A number of different chromogens have been proposed for use in this procedure. A commonly employed chromogen is diamino benzidine.
The reagents of the prior art which have been applied in the Western Blot procedure have suffered from very short shelf lives. The co-substrate hydrogen peroxide which if present in the reagent together with the chromogen substrate is per se an oxidizing agent which has the inherent capacity for converting the chromogen in a short time to its colored derivative.
The chromogen substrates of the prior art also are relatively insensitive and carcinogenic.
It is the general purpose of the present invention to provide a non-carcinogenic reagent which has a shelf life of six months or more when mixed with its co-substrate hydrogen peroxide, and which, in addition, is highly sensitive to reaction, being converted rapidly to an intensely colored, highly visible, insoluble product when contacted with even very small amounts of the peroxidase component of the Western Blot test strip.
This result is achieved by inclusion in the reagent of a proportion of a unique chromogen of the general formula ##STR1## and the benzene ring is substituted at the 5 or 7 position with an amino group.
More explicitly, the chromogens useful in the reagents of the invention comprise members of the following group:
5 amino-indole PA1 7 amino-indole PA1 5 amino-benzimidazole PA1 7 amino-benzimidazole PA1 5 amino-benzothiazole PA1 7 amino-benzothiazole PA1 5 amino-benzoxazole PA1 7 amino-benzoxazole PA1 5 amino-indazole PA1 7 amino-indazole
These materials have the unique quality of stability in the presence of hydrogen peroxide so that the reagents of the invention remain stable over periods of several months, thus rendering them suitable for commercial application. They also are non-carcinogenic. Still further, they are extremely sensitive to the presence of minute quantities of peroxidase and develop in the presence of hydrogen peroxide an intense coloration of the bands of the test strip if disease antibodies are present on it.
The underlying basis for the special properties of the above group of chromogens is determined by the fact that they all comprise nitrogen compounds of which a nitrogen atom is a component of a heterocyclic ring. This imparts stability to the chromogen.
When placed in contact with the antibody-bound peroxidase in the presence of hydrogen peroxide, the chromogen first is converted to a quinimine monomer characterized by the presence of positive charges at the 4, 6 and 7 carbon atoms in the case of the 5-amino chromogens, and at the 4, 5 and 6 positions in the case of the 7-amino chromogens. This characteristic, in turn, promotes polymerization of the monomer to a highly colored random polymer linearly and cross-linked at the 4, 6 and 7 carbon atoms in the one case, and at the 4, 5 and 6 carbon atoms in the other. The resulting polymer is water insoluble because of its molecular content of a large member of hydrophobic aromatic rings.
In either case, when carrying out the Western blot test described above, the highly colored polymeric product deposits at the site of the co-valently bound enzyme, thus designating specifically the position of the bound antibody on the test strip.